Lung transplantation is a lifesaving option for veterans with end-stage lung diseases, in particular idiopathic pulmonary fibrosis (IPF). Veterans appear to be disproportionately affected by IPF, a disease that has been described as early aging of the lung. IPF is usually fatal unless the lungs are replaced by transplant. Even following lung transplantation median survival is less than six years, limited primarily by chronic lung allograft dysfunction (CLAD). Emerging data suggest that telomeres, the nucleoprotein caps that protect chromosomes during cellular replication, are involved in IPF, but it is unknown whether telomeres also play a role in CLAD. Were that to be the case, the same pathophysiology that necessitated transplant might also underlie its failure. Our own preliminary data show that impaired telomeres in peripheral blood of lung allograft donors are linked to decreased survival in lung allograft recipients. We also have found that telomere dysfunction in airway stem cells is sufficient to induce the pathologic hallmarks of CLAD in an experimental murine model. In humans, airway progenitor cells proliferate and differentiate to restore airway epithelial integrity following injury. Thus, telomere dysfunction could lead to airway epithelial cell progenitor failure, resulting in denuded airways that are subsequently replaced by fibrotic tissue. With the support of this Merit Award, we will test the innovative hypothesis that telomere dysfunction leads to CLAD. In Study Aim 1, we will evaluate the associations between telomere genetic variants and CLAD in a large established multi-center cohort of lung transplant recipients. Common genetic variants resulting in short telomeres will be sequenced from donor cells, and telomere length will be determined by quantitative PCR. We will use adjusted Cox proportional hazards models to evaluate the links between donor telomere length or genotype and post-transplant survival time. Novel genotypic associations with telomere dysfunction will be validated in vitro. These findings will help distinguish the contributions of innate and acquired telomere dysfunction to poor post-transplant outcomes. Study Aim 2 will test the association between short allograft epithelial cell telomeres and CLAD-free survival in a longitudinal cohort. Epithelial telomere lengths will be determined by fluorescence-in situ hybridization with a telomere-specific probe (Telo-FISH) on endobronchial and transbronchial biopsy tissues. We will test the association between telomere length and CLAD-free survival using adjusted Cox models and examine transcriptomic sequelae of telomere dysfunction. In Study Aim 3, we will determine whether airway epithelial cell injury is associated with allograft telomere shortening and epigenetic aging in a prospectively enrolled cohort of lung transplant recipients. Early allograft injury will be assessed clinically by the presence of primary graft dysfunction (PGD). We also quantify allograft epithelial cell injury from recipient plasma cell-free DNA using next-generation bisulfite sequencing to enumerate donor-specific polymorphisms and cell-type specific DNA methylation patterns. We will test for associations between PGD and cell-free DNA measurements of allograft injury with telomere-based and epigenetic metrics of allograft aging using adjusted linear models. These findings will shed light on the longstanding question of how acute lung injury and inflammation develop into chronic fibrosis. Further, these studies could establish that CLAD is an evoked phenotype in which telomere dysfunction leads to impaired epithelial responses to chronic transplant-associated airway injury. Overall, this proposed investigation has the potential to reshape our conceptual understanding of CLAD and lead to novel biomarkers that could inform cutting edge therapeutic interventions. This would be new paradigm, potentially transforming our approach CLAD and thus improving outcomes for veterans with end- stage lung disease.